Aberrant Methylation-Mediated Suppression of APAF1 in Myelodysplastic Syndrome.

Background: Myelodysplastic syndromes (MDSs) include a diverse group of clonal bone marrow disorders characterized by ineffective hematopoiesis and pancytopenia. It was found that down regulation of APAF1, a putative tumor suppressor gene (TSG), leads to resistance to chemotherapy and disease development in some cancers. In this study, we investigated the relation of APAF1 methylation status with its expression and clinicopathological factors in myelodysplastic syndrome (MDS) patients. Materials andMethods: Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) was employed in studying the methylation of CpG islands in the APAF1promoter region in MDS. Gene expression was analyzed by using real time RT-PCR. Results: 42.6% of patient samples were methylated in promoter region of APAF1analyzed, while methylation of the gene was not seen in controls (P<0.05). Methylation of APAF1was significantly associated with the suppression of its mRNA expression (P=0.00). The methylation status of APAF1in advanced-stage MDS patients (80%) was significantly higher than that of the early-stage MDS patients (28.2%) (P=0.001). The difference in frequency of hypermethylatedAPAF1 gene was significant between good (37.5%) and poor (85.71%) cytogenetic risk groups (P=0.043). In addition, a higher frequency of APAF1hypermethylation was observed in higher-risk MDS group (69.2%) compared to lower-risk MDS group (34.14%) (P=0.026). Conclusion: Our study indicated that APAF1hypermethylation in MDS was associated to high-risk disease classified according to the IPSS, WHO and cytogenetic risk.


INTRODUCTION
MDSs, a heterogeneous group of clonal stem cell disorders, are characterized most commonly by a hypercellular bone marrow, cytopenias and progression to acute myeloid leukemia (AML) 1,2 .Hypermethylation of CpG islands located in the promoter regions is known to be a frequent and early event in carcinogenesis that regulates gene expression, including expression of tumor suppressor genes (TSGs) and apoptotic genes. 3,4 There is increasing evidence that aberrant methylation of DNA is one of the underlying mechanisms in pathogenesis of MDS and leukemic transformation. 5 It has been shown that deregulation of apoptosis results in irregular cell survival and has been implicated in the development of cancer. Apoptotic protease activating factor1 (APAF1),a putative TSG, encodes one of the important cytoplasmic proteins in DNA damage -induced apoptosis and is therefore essential for tumor suppression. 6,7 In apoptotic cascade, cytochrome C released from the mitochondria in response to cell death stimulus is bound to APAF1in the cytosol. APAF1 in association International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir with ATP is released from auto-inhibited state and would be bound to procaspase. 9 Thus, ATP binding allows oligomerization of APAF1, which is necessary for auto-activation of caspase-9, successive activation of downstream procaspases and eventual programmed cell death (apoptosis). [8][9][10] In addition, APAF1 is recognized as an essential downstream effector of p53-mediatedapoptosis. 11,12 Given the role of APAF1 in apoptosis, it seems that its inactivation plays a role in oncogenic transformation and drug resistance. [13][14][15] In this study, we aimed to elucidate methylation status of CpG islands in the APAF1 promoter region in MDS patients. In addition, the association between the methylation status of APAF1 and its expression with clinicopathological variables of patients was also evaluated. To conclude then, in this study we have demonstrated elevation in hypermethylationofAPAF1 and subsequently suppression of its expression in advanced-stage MDS, indicating that APAF1 might be involved in disease progression. criteria. Clinical and demographic data were summarized in Table 1.

Correlation between Gene Methylation and Expression in MDS Patients
Quantitative Real-time RT-PCR was done to determine whether the promoter methylation was related to mRNA downregulation.APAF1 promoter methylation was shown to be inversely correlated with the mRNA expression in patients (P=0.00).There was a significant difference in the delta Ct of the APAF1 mRNA between patients and normal subjects/controls using the two-tailed t-test (P= 0.007). Delta Ct average was significantly greater in patients with advanced-stage MDS (average delta Ct 3.6) compared with early-stage MDS (average delta Ct 2.6) or controls (average delta Ct 1.6). The difference in the delta Ct between advanced-stage MDS and controls was statistically significant (P=0.001).Gene expression average in early-stage MDS was higher than that in advancedstage MDS and this difference was statistically significant (2.62 vs. 0.46, P=0.03). The highest and the least average in APAF1 fold change was observed in RCMD and RAEB-1, respectively (4.14 vs. 0.11, respectively). We discovered that APAF1expressionfoldchangewashigherinlow-risk MDS than that in high-risk MDS (2.23 vs. 1.69, respectively), but it was not statistically significant (P=0.535). In further analysis, the correlation between gene expression and IPSS score was statistically significant (P=0.02). No significant differences were observed between APAF1 fold change and WBC, ANC, platelets, Hb, age, SF and LDH levels, cytogenetic risk groups and IPSS-R in MDS patients (P>0.05).

APAF1Promoter Was Hypermethylated in Patients with MDS DNA methylation was measured with the MS-HRM.
Standard curves with commercial controls were drawn for validation of HRM ( Figure 1).

DISCUSSION
Apoptosis is one of the main pathways often deregulated in MDS. 17 There is increasing evidence that during early course of MDS disease, increased apoptosis is associated with decreased cell survival, while during later phases, the overexpression of pro survival proteins, decreases apoptotic rate of bone marrow cells and promotes the possibility of the evolution of MDS syndromes to AML. 17,18 The aberrant methylation of genes involved in the apoptotic pathway is poorly defined in MDS. When aberrant methylation occurs in the promoter of genes that suppress tumorigenesis, it can lead to the resistance to apoptosis and progression of cancer 19 .Since theAPAF1gene, a TSG, is rarely mutated, promoter hyper methylation appears to be the mechanism underlying its inactivation. 20 In this study, we examined expression of APAF1 in MDS and found that APAF1 mRNA level was diminished in 53.7% of MDS cells. Our data indicated a higher level of APAF1 transcripts in lowrisk MDS compared to the high-risk disease as categorized based on WHO and IPSS (P<0.05). No difference was observed when the groups were compared according to the cytogenetic risk score (P>0.05). Consistent with other investigations, a decreased expression of APAF1 in high-risk MDS may be indicative of a role for APAF1 in MDS progression or lower levels of apoptosis in high-risk MDS compared to low-risk MDS. It has been shown that increased expression of APAF1in low-risk disease and its positive correlation with the apoptotic rate may be indicative of APAF1rolein the pathophysiology of MDS. 21 Aberrant DNA methylation has been associated with downregulation of genes. 22,23 The relationship between prognosis and DNA methylation has been studied in MDS on a single candidate gene or combinations of multiple genes. For instance, a study of patients with MDS showed that concordant methylation of multiple genes predicts poor prognosis and risk of leukemia transformation. 24 Our analysis of methylation status of APAF1gene promoter in patients with MDS showed thatAPAF1hypermethylation frequency was 42.6%. In this study, we presented evidence for elevation of APAF1methylationin MDS patients during the progression from low-risk (28.2%) to the high-risk disease (80%). An association of methylation-associated silencing of APAF1 expression with higher IPSS-R score and advanced-stage MDS was recorded. Our results suggest that APAF1 may be implicated in the acquisition of a more aggressive phenotype in MDS. Cytogenetic analysis is one of important risk factors for predicting leukemic evolution. 25 In addition, a recent study provides new information about the role of cytogenetic analysis in diagnosis, prognosis and follow-up of patients with hypocellular primary MDS. 26 In our analysis, a correlation with chromosomal aberrations was elicited for APAF1 promoter methylation. These results may indicate that hyper methylation may contribute to the leukemogenesis. LDH is a useful prognostic parameter in several hematological malignancies. [27][28][29] We discovered the association betweenAPAF1 hypermethylation and initial LDH level to a statistically significant extent. In further analysis, there was a strong correlation between cytogenetic risk categories, MDS subgroups and IPSS-R with level of LDH activity. High LDH level remained a significant adverse prognostic factor for high-risk patients. The results showed that APAF1 promoter hypermethylation was correlated closely with the loss of APAF1 mRNA expression, indicating that function of this gene may recover following demethylation. The present study, along with previous reports 30 , determines APAF1 as another target of methylation silencing. The evidence has been shown that demethylation treatment can restore expression of APAF1 at both mRNA and protein levels and, therefore, activate  20 In the present study, following statistical analysis, the expression and hypermethylation of APAF1 was not significantly correlated with the age, gender, hematologic variables and SF of the patients (P>0.05).
In conclusion, the present study is consistent with some observations in oncogenesis that have identified the loss of APAF1 as a substantial characteristic in development of tumor. With the increased progression of MDS, the expression of APAF1 mRNA tends to decrease. Gene silencing following methylation is an important epigenetic mechanism of gene down-regulation. Furthermore, our results imply that hypermethylation is associated to highrisk disease as classified according to the IPSS, WHO, and cytogenetic risk. Therefore, APAF1 may serve as prognostic indicator in advanced-stage MDS. Our results also confirm baseline LDH as a significant prognostic marker.

CONCLUSION
In summary, the present study is consistent with some observations in oncogenesis that have identified the loss of APAF1 as a substantial characteristic in development of tumor. With the increased progression of MDS, the expression of APAF1 mRNA tends to decrease. Gene silencing following methylation is an important epigenetic mechanism of gene down-regulation. Furthermore, our results imply that hypermethylation is associated to high-risk disease as classified according to the IPSS, WHO, and cytogenetic risk. Therefore, APAF1 may serve as prognostic indicator in advanced-stage MDS. Our results also confirm baseline LDH as a significant prognostic marker.

ACKNOWLEDGMENT
This work was supported by Iran University of Medical Sciences (IUMS) and Iran National Science Foundation (INSF) grants. We would like to thank our colleagues from Hematology, Oncology and Stem cell Transplantation Research Center in Shariati Hospital who provided patient samples that greatly assisted the research. We are also pleased to acknowledge the cooperation of patients for donating their samples.